15.11.2013

Diabetes Fact #1: In 2010, there were an estimated 346 Million people suffering from diabetes worldwide according to the WHO.
Insulin dependent or Type 1 diabetes is caused by insufficient amounts of insulin being produced.
In the non-insulin dependant diabetes or Type 2 Diabetes, the body produces insulin, but uses it ineffectively.
Diabetes Fact #5: Diabetes has many health consequences, especially when blood sugar levels are not kept under control.
Diabetes Fact #8: Treating and living with diabetes, is very costly, both to yourself in terms of lost work, medical expenses and much more, but also to the State in the form of health care and Disability Grants.
It is just as important to make up your mind you are not going to be part of these diabetes statistics while you are healthy. DisclaimerThe contents on this blog are personal opinions, experiences and research and should not be accepted and implemented without further research or consulting your physician.
In Diabetes Type 1 the body is not producing insulin, while in Diabetes Type 2 the cells are not responding properly to the insulin, and there is not enough insulin being produced.
When ever food enter in our body ,Food get converted into the Glucose and because of insulin it enter and adsorb by the our body so the insulin is the main part and factor by which our body can absorb the glucose.
Insulin, a hormone, is produced by Beta cells in the Islets of Langerhans, which are in the pancreas. So if you have diabetes then your body or bloodstream will not absorb Glucose properly or not at all absorb so this activity resulted high amount of Glucose and one the amount of glucose got high level than this situation called hyperglycemia.
When the cell of body does not respond to insulin than this situation is called Diabetes Type 2.
So when body is not able to get proper energy and continuously increasing the level of Glucose than it a time people to get worry and rush to your doctor.
So basically so cannot reduce Diabetes Type 1 through exercise because the beta cell has already destroyed. The major quantity of diabetes patient has Diabetes Type 2 (Approx 85 %) and patient usually seems over weight and unfit.This kind of diabetes comes late in the life and it is very uncommon to find Diabetes Type 2 in 20s age people.
Guys here we have written what we can but if you and your dear one is suffering from diabetes type 1 or diabetes type 2 than you must rush towards doctors and for you later on we will also publish the home remedies to cure diabetes. From 1993 to 2009, trends in age-adjusted hospital discharge rates for nontraumatic LEA among the diabetic population were similar by levels of LEA.
Diabetes is on the rise in Australia and the rest of the world and has reached epidemic proportions. Feeling tired and lethargic, always feeling hungry, having cuts that heal slowly, skin infections, blurred vision, gradual weight gain, mood swings, headaches and dizziness.
You are more chance of becoming diabetic if you have a family history of diabetes or if you are over 55 years of age – as the risk increases as we age.
There are so many benefits to strength training: increased muscle strength and power, increased muscle size and endurance, reduction in body fat, increased bone mineral density, increased metabolic rate, lower blood pressure, increased sense of well being and self esteem… do I need to keep going? Strength training programs should be designed by qualified trainers (and supervised where possible) and and be reviewed regularly so that weights are gradually increased.
If you think you know someone who might be at risk of type 2 diabetes, their GP is the best place to start. Science, Technology and Medicine open access publisher.Publish, read and share novel research.
DNA Immunotherapies for Type 1 DiabetesAlice Li1 and Alan Escher2[1] Loma Linda University, Loma Linda, California, USA[2] SEKRIS Biomedical Inc., Redlands, California, USA1.
Beta-Cell Function and FailureSoltani Nepton1[1] Physiology Department, Faculty of Medicine, Hormozgan University of Medical Science, Iran1.
Russ HA, Sintov E, Anker-Kitai L, Friedman O, Lenz A, Toren G, Farhy C, Pasmanik-Chor M, Oron-Karni V, Ravassard P, Efrat S. Soltani N, Qiu H, Aleksic M, Glinka Y, Zhao F, Liu R, Li Y, Zhang N, Chakrabarti R, Ng T, Jin T, Zhang H, Lu WY, Feng ZP, Prud'homme GJ, Wang Q. In USA the Hispanic Puerto Rican group have the highest prevalence at 13.8% and the non-Hispanic white group the lowest at 7%.
People suffering from diabetes cost the Government more than twice as much as people without diabetes. Although they can be depressing at first sight, they can also be the motivation to do something different to what we have always done in terms of health.
Centers for Disease Control and Prevention (CDC), National Center for Health Statistics, Division of Health Interview Statistics, data from the National Health Interview Survey. You are also at risk if you are over 45 years of age and are overweight or have high blood pressure or you are over 35 years of age and are from an Aboriginal or Torres Strait Islander, or Pacific Island, Indian subcontinent or Chinese cultural background. Particularly for people with diabetes, strength training and increased muscle mass means more uptake of insulin into the muscle, (and therefore less glucose floating around in the bloodstream causing trouble.) Exercise increases the amount of insulin receptors released and an increase in muscle cells from strength training means more opportunities for insulin to bind to those receptors and move glucose from the blood into the cells.
Contrary to the popular belief that exercise should be light, research shows that type 2 diabetics, with no other contraindications, will experience the most benefit from moderate to hard weight training sessions. The table summarizes plasmid DNA based immunotherapies under two categories: Gene therapies and DNA vaccines.
Prevention of beta cell dysfunction and apoptosis by adenoviral gene transfer of rat insulin-like growth factor 1.
ICV brain glibenclamide suppresses counterregulatory responses to brain glucopenia in rats: evidence for a role for brain KATP channels in hypoglycemia sensing. The function of CA(2+) channel subtypes in exocytic secretion: new perspectives from synaptic and non-synaptic release. Prevention of beta cell dysfunction and apoptosis activation in human islets by adenoviral gene transfer of the insulin-like growth factor I.
Beta-cell dysfunction and insulin resistance in type 2 diabetes: role of metabolic and genetic abnormalities. Effect of the administration of Psidium guava leaves on lipid profiles and sensitivity of the vascular mesenteric bed to phenylephrine in STZ-induced diabetic rats. Insulin-Producing Cells Generated from Dedifferentiated Human Pancreatic Beta Cells Expanded In Vitro. The prevalence of diabetes is increasing rapidly especially in developing countries like China and India. In poorer countries, specially if they copy the more industrialized lifestyle, where there are often no facilities for diagnosis or treatment, the numbers could be much higher. More than half of all diabetic patients suffer from neuropathy in some form, usually in the limbs. Data computed by personnel in the Division of Diabetes Translation, National Center for Chronic Disease Prevention and Health Promotion, CDC.
Without going into too much detail, diabetes is essentially a condition where the body is not able to regulate its blood glucose levels.
Plasmid DNA immunotherapy for type 1 diabetes can be divided into two categories: DNA vaccines and gene therapies. Furthermore, similar to other autoimmune diseases, allergies and asthma, the incidence of type 1 diabetes is on the increase at an alarming rate in industrialized countries for unknown reasons.
Beta cells (?-cells)Beta cells are a type of cell in the pancreas located in the so-called islets of Langerhans. Program and abstracts of the 62nd Scientific Sessions of the American Diabetes Association, 14(18), 2002; San Francisco, California. Program and abstracts of the 62nd Scientific Sessions of the American Diabetes Association, 17, 2002; San Francisco, California. Program and abstracts of the 62nd Scientific Sessions of the American Diabetes Association, 14(18), 2002, San Francisco, California.
Effect of oral magnesium sulfate administration on blood pressure and lipid profile in streptozocin diabetic rat.Eur J Pharmacol. Type 2 diabetes occurred traditionally in older people, but the worrying fact now is that younger people and even children are suffering from Type 2 diabetes.
But I don’t want to freak anyone out here, what I do want to talk about is how weight training can help with management of diabetes.
Plasmid DNA can be delivered using different routes, for example, the intradermal and intramuscular routes. Information released by the American Diabetes Association shows 23% and 21% increased rates of type 1 and 2 diabetes, respectively, from 2001 to 2009.
They make up 65-80% of the cells in the islets.The Islets diameter is about 50 to 300 micrometers. Program and abstracts of the 62nd Scientific Sessions of the American Diabetes Association; 14(18), 2002, San Francisco, California. Extracts on insulin release from in situ isolated perfused rat pancrease in newly modified isolation method: the role of Ca and K channels. If diabetes statistics continue at their present levels of acceleration, the WHO predict diabetes will be the 7th leading cause of death in the entire world within 20 years.
Of these, nearly a quarter of a million were children and young people under the age of 20 years. The consens now is that Type 2 diabetes occurs predominantly in people with incorrect eating habits, obesity and those who lack exercise. Glucose (sugar) is carried in the blood and uses a hormone called insulin (produced by the pancreas) to convert into energy for use by the cells of our muscles. The increase in incidence of type 1 diabetes is especially apparent in young children and has generated an urgent need for novel treatments that can safely control diabetes-causing inflammation, and alleviate the need for administration of exogenous insulin.
In people with diabetes insulin is no longer produced, or not produced in enough amounts so the glucose remains in the blood stream. Indeed, type 1 diabetes has been treated for almost a century in the same fundamental manner using daily insulin injections. DNA vaccine immunotherapiesPlasmid DNA vaccine-based immunotherapy is a promising therapeutic field for treatment of type 1 diabetes.
Although it is a life-saving treatment and one of the most remarkable accomplishments of medicine, administration of exogenous insulin is still not a cure because it does not address the underlying autoimmunity that targets insulin-secreting beta cells. These cells are surrounded by alpha cells that secrete glucagon, smaller numbers of delta cells that secrete somatostatin, and PP cells or F cells that secrete pancreatic polypeptide. Because it is currently impossible to mimic regulation of physiological insulin levels faithfully, many type 1 diabetic individuals receiving standard of care are exposed to acute and chronic complications that cause increased morbidity and mortality. Nevertheless, although beneficial effects were observed, it is clear that efficacy must be significantly improved. All of the cells communicate with each other through extracellular spaces and through gap junctions.
As a result, there have been intense efforts to develop immunotherapies that can eliminate or at least alleviate the need for exogenous insulin. Improving efficacy will be likely dependent on the ability to modulate both the innate immune system, through activation of tolerogenic antigen-presenting cells like dendritic cells, and the adaptive immune system, through activation of various populations of regulatory cells.
This arrangement allows cellular products secreted from one cell type to influence the function of downstream cells.
In this case the goal is to arrest pathological autoimmunity that destroys beta cells so that the cells can regain function, and possibly proliferate and regenerate.
DNA vaccines are particularly well positioned to achieve this goal because plasmid DNA is information-based, and can encode molecules that affect the immune system in different manners. As an example, insulin secreted from beta cells can suppress glucagon secretion.A neurovascular bundle containing arterioles and sympathetic and parasympathetic nerves enters each islet through the central core of beta cells.
The challenge is to identify which combination of functions should be delivered together with a pancreatic autoantigen to treat disease with maximum efficacy and safety.Several beta cell autoantigens have been tested in mice for induction of immune tolerance by DNA vaccines and will be discussed in this section. The arterioles branch to form capillaries that pass between the cells to the periphery of the islet and then enter the portal venous circulation.2.
The first path relies on different forms of systemic suppression of inflammation that inhibit effector T lymphocytes in a non-specific manner.
Immune mechanisms associated with the therapeutic effects of DNA vaccines can be complex because of the variety of cells that can process the information encoded by plasmid DNA.
Beta cells functionsInsulin is synthesized as preproinsulin in the ribosomes of the rough endoplasmic reticulum in the beta cells (fig 1).
Serious side effects associated with the use of systemic immunotherapies are increased risks of cancer and infection resulting from the decreased activity of effector cells involved in beneficial destructive immune responses against cancer cells and pathogens. Preproinsulin is then cleaved to proinsulin, which is transported to the Golgi apparatus where it is packaged into secretory granules located close to the cell membrane.
These side effects have been observed with broadly acting immunosuppressants used to prevent organ transplant rejection, which have been also investigated for treating type 1 diabetes [1, 2]. Insulin DNA vaccinesThus far, the only DNA vaccine that has been tested in both preclinical and clinical trials is a plasmid DNA construct coding for intracellular proinsulin, which is a partially processed non-functional form of insulin. Proinsulin is cleaved into equimolar amounts of insulin and C-peptide in the secretory granules. Serious side effects can also be seen, albeit to a lesser degree, with more specific agents like antibodies that target specific molecules involved in inflammation [3, 4]. Insulin is not only the hormone produced by beta-cells that controls carbohydrate and fat metabolism in the body, it is also a main target autoantigen in autoimmune diabetes and the presence of anti-insulin autoantibodies can be an indication of disease initiation [59]. The process of insulin secretion involves fusion of the secretory granules with the cell membrane and exocytosis of insulin, C-peptide, and proinsulinInsulin is a hormone that controls the blood glucose concentration.
DNA vaccines coding for different forms of insulin have been investigated for type 1 diabetes immunotherapy since the late 1990’s.
The liver maintains the base-line glucose level, but the beta cells can respond quickly to spikes in blood glucose by releasing some of its stored insulin while simultaneously producing more. This path is thought to be safer because it aims to induce a regulatory immune response that targets the inflamed islets. The response time is very quick.Figure 1Mouse pancreatic islet as seen by light microscopy. Here the goal is to manipulate endogenous immune mechanisms of homeostasis that can re-establish some form of tolerance to the chosen autoantigen, as well as to other neighboring beta cell autoantigens through a mechanism known as “by-stander suppression” [5]. The DNA vaccine induced insulin B-chain specific CD4+ T regulatory cells that secreted interleukin-4, and locally reduced autoreactive activity of cytotoxic T lymphocytes in the pancreatic draining lymph nodes. Accordingly, it is anticipated that pathological autoimmunity and inflammation of islets can be stopped in an organ-specific manner that does not impair the immune system. Further work showed that co-delivery of interleukin-4 was required to prevent diabetes onset in male nonobese diabetic mice [61]. Glucagon is labeled in red and the nuclei in blueApart from insulin, beta cells release C-peptide, a consequence of insulin production, into the bloodstream in equimolar amounts. In this chapter, we will review how plasmid deoxyribonucleic acid (DNA) has been used as an immunotherapeutic vector platform to treat type 1 diabetes through each immunotherapeutic path.
Two isoforms of insulin are synthesized in rodent animals, insulin I in islets and insulin II in both islets and thymus while humans have only one form of insulin. For the purpose of this review, we have called the first path “gene-based immunotherapy”, meaning that plasmid DNA does not encode a known autoantigen, and the second path “DNA vaccine immunotherapy” meaning that a beta cell autoantigen is encoded by plasmid DNA (Figure 1). The pancreatic beta cells synthesize proinsulin before converting it to functional insulin. In other words, gene-based immunotherapy relies on the inherent function of a product encoded by plasmid DNA that can in turn affect cell function. In that regard, intranasal delivery of plasmid DNA encoding mouse proinsulin II together with injection of an anti-CD154 (also named CD40 ligand) antibody to prevent T cell activation was reported to prevent type 1 diabetes in nonobese diabetic mice [62]. Amylin's metabolic function is now somewhat well characterized as an inhibitor of the appearance of nutrient [especially glucose] in the plasma. In contrast, DNA vaccine immunotherapy relies on the tolerogenic immune response induced directly by the autoantigen after its processing by immune cells.

Delivery of 300 microgram DNA and 50 microgram antibody over a 2-week interval at 4 weeks of age synergistically prevented diabetes, reducing disease incidence from 100% diabetic down to 0% in 40-week old mice. As we shall see, some DNA vaccine immunotherapies have also a gene-based immunotherapy component that acts as a molecular adjuvant to promote tolerogenic immune responses.
Whereas insulin regulates long-term food intake, increased amylin decreases food intake in the short term.GABA (? amino butyric acid) is produced by pancreatic beta cell.
Nevertheless, in all cases synthesis of the molecule encoded by plasmid DNA, which is almost always a protein, starts after delivery of the plasmid DNA and its uptake by cells.
However, delivery of the DNA vaccine alone did not reduce diabetes incidence, even though it could induce T regulatory cells and reduce insulitis. GABA released from beta cells can act on GABA Areceptor in the ? cells, causing membrane hyperpolarization and hence suppressing glucagon secretion. Plasmid DNA has several notable advantages compared to other vectors and therapeutic molecules.
Another report has shown that co-delivery of 50 microgram plasmid DNA encoding human proinsulin together with 100 microgram insulin peptide twice over a 2-week interval could prevent diabetes until 24 weeks of age in 6 week old nonobese diabetic mice. An impaired insulin-Akt-GABAA receptors glucagon secretory pathway in the islet may be an underlying mechanism for unsuppressed glucagon secretion, despite hyperglycemia, in diabetic subjects.
For example, it consists of relatively low molecular weight circles of double stranded DNA that can be readily isolated from bacteria in a generic and cost-effective manner.
Some studies demonstrated that beta cells also express GABA A receptors, forming an autocrine GABA signaling system. In addition, plasmid DNA permits rapid turnaround when developing new candidate products, refrigeration-free storage, and synthesis over time of a chosen antigen in its native conformation. Results also indicated induction of CD4+CD25- islet specific T regulatory cells producing transforming growth factor-beta only in the co-immunization group.In another study, a DNA vaccine encoding proinsulin and pancreatic regenerating (Reg) III protein resulted in a significant reduction of hyperglycemia and diabetes incidence with increased serum insulin in a streptozotocin- induced mice model [64]. However, the role of this autocrine GABA signaling in the regulation of beta cell functions remains largely unknown. Furthermore, plasmid DNA can be given in repeat doses within short periods of time without inducing an immune response to vector and other side effects.
Zinc can keep insulin molecules together in the beta cells.Beta cells must have zinc to function.
Although they may sometime have pro-inflammatory properties that can be detrimental to controlling inflammation, these particles have been used to induce immune tolerance with plasmid DNA. For example, chitosan-DNA nanoparticles encoding an ovalbumin antigen are tolerogenic when delivered orally [9].
Indeed, route of delivery can play a significant role in the type and strength of immune responses induced by DNA vaccines in animal models [10, 11]. Mechanisms of insulin secretion from beta cellsThe secretion of insulin from pancreatic beta cells is a complex process involving the integration and interaction of multiple external and internal stimuli. In humans, two microgram of a DNA vaccine for treatment of melanoma delivered with gold particles into skin was found to be as efficacious as 1000 microgram injected intramuscularly [12]. Thus, nutrients, hormones, neurotransmitters, and drugs all activate or inhibit insulin secretion. These results illustrate the significant impact that choice of route and method of delivery of a DNA vaccine can have not only on efficacy, but also on cost of treatment. The primary stimulus for insulin release is the beta-cell response to changes in glucose concentration. Other delivery routes like intravenous, nasal, and sublingual have also been investigated [13].
Post delivery, expression of coding sequences in plasmid DNA results in significant levels of protein production that may persist for six weeks and longer without serious side effects in human patients [14, 15]. First-phase insulin release occurs within the first few minutes after exposure to an elevated glucose level; this is followed by a more permanent second phase of insulin release.
Of particular importance is the observation that first-phase insulin secretion is lost in patients with type 2 diabetes. DNA vaccines and other gene-based vaccines belong to a third generation of vaccines after live and attenuated whole organism vaccine and recombinant protein vaccines. In the K (ATP) channel-dependent pathway, glucose stimulation increases the entry of extrinsic Ca2+ through voltage-gated channels by closure of the K (ATP) channels and depolarization of the beta cell membrane. These vaccines can be used to either prevent (prophylactic vaccine) or treat (therapeutic vaccine) disease depending on their potency, in which prevention is generally easier to achieve than treatment. Recent reports of beneficial results in different clinical trials using delivery of autoantigens indicate that DNA vaccination is reaching a stage where we are likely to see accelerated development of a therapeutic future for vaccines targeting a variety of autoimmune diseases.
While in the GTP-dependent pathway, intracellular Ca2+ is elevated by GTP-dependent proteins and augments the Ca2+-stimulated release.
In the case of type 1 diabetes, early results using a DNA vaccine encoding insulin have shown promise in humans. Secretagogues and insulin secretion inhibitors act at intermediate steps of these signaling pathways and influence the process of insulin exocytosis.
In addition, DNA vaccines encoding human heat shock protein 60 and glutamic acid decarboxylase 65 have also shown efficacy in preclinical trials and are reviewed in this chapter. Several researchers have investigated this intricate mode of known secretagogue action using isolated islets as an in vitro model. In contrast to DNA vaccine immunotherapy, gene-based immunotherapy involves delivery of genetic material by a plasmid vector into a cell, tissue or organ with the aim of improving the clinical status using the function of the encoded product, instead of its properties as an antigen. To quote a few; imidazoline antagonists of alpha 2-adrenoreceptors increase insulin release in vitro by inhibiting ATP-sensitive K+ channels in pancreatic beta cells. Gene-based immunotherapy includes delivery of anti-inflammatory cytokines, chemokines, and other factors to modulate the activity of immune cells [16, 17].2. Some researchers have evaluated the properties of sulphonylurea receptors (SUR) of human islets of Langerhans. Gene-based immunotherapiesSeveral pre-clinical trials have used plasmid DNA-based gene therapies in experimental models of autoimmune type 1 diabetes. They studied the binding affinity of various oral hypoglycaemic agents to the receptor and also tested insulinotropic action of the drugs on intact human islets. These strategies involve plasmid DNA designed to weaken pre-existing beta-cell autoimmunity through delivery of anti-inflammatory cytokines, chemokines, and other immune cell manipulating agents. This binding potency order was parallel with the insulinotropic potency of the evaluated compounds.
Some investigators have shown an insulinotropic effect of Triglitazone (CS-045) and have shown its mode of action to be distinct from glibenclamide (a sulphonylurea drug). Cytokine gene therapiesCytokine gene therapies are strategies that use engineered plasmid DNA to produce therapeutic immune cytokines, which are a group of immune active molecules secreted by different cells of the body. A-4166, a derivative of D-phenylalanine, evokes a rapid and short-lived hypoglycaemic action in vivo.
Some of these cytokines are considered beneficial for the suppression of autoimmunity, and thus are applied to disease models to reduce clinical symptoms and improve therapeutic effects.
Studies of animals with spontaneous autoimmune diabetes have revealed that an important group of autoreactive T cells that mediates islet beta-cell destruction belongs to the T helper-1 type effector cell subset, and produces cytokines like interleukin-2 and interferon-gamma.
Some studies showed S21403, a meglitinide analogue to be a novel insulinotropic tool in the treatment of type 2 diabetes, as it affected cationic fluxes and the drugs secretary responses displayed favourable time course of prompt, and not unduly prolonged, activation of beta cells. On the other hand, regulatory T cells that control effector cells can secrete interleukin-4, interleukin-10, and transforming growth factor-beta. Some studies demonstrated that tetracaine (an anaesthetic) stimulates insulin secretion by release of intracellular calcium and for the first time elucidated the role of intracellular calcium stores in stimulus-secretion coupling in the pancreatic beta cells.
One of the earliest applications of cytokine-engineered plasmid DNA was gene-gun delivery of murine interleukin-4 to prevent spontaneous type 1 diabetes [18]. JTT-608, is a nonsulphonylurea oral hypoglycaemic agent which stimulates insulin release at elevated but not low glucose concentrations by evoking PKA-mediated Ca2+ influx.4. The plasmid DNA was delivered as three times two microgram within 4 weeks into 3-week-old nonobese diabetic mice, which is the animal model system closest to human type 1 diabetes. The importance of KATP channelsThe KATP channels play an integral role in glucose-stimulated insulin secretion by serving as the transducer of a glucose-generated metabolic signal (ie, ATP) to cell electrical activity (membrane depolarization). Type 1 diabetes incidence was reduced from 90% in controls to 20% at 34 weeks of age, and was associated with T helper-2 type immune responses in the periphery and pancreas of mice. Thus, like neurons, beta cells are electrically excitable and capable of generating Ca2+ action potentials that are important in synchronizing islet cell activity and insulin release. Two other reports have shown that systemic delivery of plasmid DNA constructs coding for interleukin-4 can prevent insulitis, which is an inflammatory sign of immune cell infiltrating pancreatic islets in nonobese diabetic mice [19, 20]. In addition to being signal targets for glucose, KATP channels are the targets for sulfonylureas, which are commonly prescribed oral agents in the treatment of type 2 diabetes. For example, a report indicates that intramuscular electroporation delivery of 50 µg plasmid DNA encoding interleukin-4 accelerated spontaneous type 1 diabetes in nonobese diabetic mice [21].In addition to interleukin-4, interleukin-10-encoded plasmid DNA alone was also tested for its diabetic suppressive effects. The sulfonylurea receptor belongs to a superfamily of ATP-binding cassette proteins and contains the binding site for sulfonylurea drugs and nucleotides. The plasmid DNA was delivered intramuscularly twice for a total of 200 microgram into 3 and 5 week old female nonobese diabetic mice [22].
Although the severity of insulitis at 13 weeks of age was not improved, the incidence of diabetes was markedly reduced to 50% at 35 week old compared to 90% with control mice. These results show that the progression of autoimmune disease in mice can effectively be suppressed by intramuscular DNA injection coding for anti-inflammatory cytokines alone. Evans, MD, Yale University Medical School, New Haven, Connecticut, and colleagues have suggested that glucose sensing in the brain during hypoglycemia may be mediated by KATP channels located in brain hypothalamic neurons. Another report investigating the immune effects of interleukin-10 DNA showed that systemic intramuscular administration of 200 microgram interleukin-10 plasmid DNA could alleviate blood glucose and insulitis in a streptozotocin induced diabetic mouse model up to day 28 post injection [24].
Thus, these molecules may also serve as new therapeutic targets for the restoration of impaired hypoglycemia awareness and glucose counterregulation in type 1 diabetes.5. In this model, pancreatic interleukin-1b and tumor necrotic factor-alpha gene expression, serum interferon-gamma concentration, and the numbers of CD4+ and CD8+ lymphocytes were decreased on day 28. Voltage-dependent Ca2+ channels: Novel regulatorsExtracellular Ca2+ influx through L-type voltage-dependent Ca2+ channels (VDCC) raises free cytoplasmic Ca2+ levels and triggers insulin secretion. A similar interleukin-10 construct was modified by introducing nuclear factor kappa-B (NF-kB) binding sites into plasmid DNA to facilitate nuclear transport of the plasmid after delivery into the cell [25]. The structure of the VDCC is complex and consists of 5 subunits: alpha1, alpha2, beta, gamma, and delta units.
A single injection of 50 microgram of the plasmid using polyethylenimine as a gene carrier in 5 week old mice reduced the degree of insulitis and serum glucose levels in 100% of mice compared to 40% of the control mice at 32 weeks of age. The alpha subunit constitutes the ion-conducting pore, whereas the other units serve a regulatory role. These results illustrate how plasmid DNA can be easily modified in a generic manner to improve therapeutic efficacy.As mentioned previously, nanoparticle technology has been used to condense plasmid DNA into nanometer-size complexes to improve delivery.
Previous work has identified that isoforms of alpha1 subunits interact with exocytotic proteins. An interleukin-10 encoding plasmid DNA was assembled into a cationic nanoparticle complex, and a single dose of 50 microgram DNA was delivered intramuscularly into streptozotocin-induced diabetic mice [26].
More recently, using the yeast hybrid screening method, a novel protein, Kir-GEM, interacting with the beta3 isoform of the VDCC, has been identified by Seino and colleagues. Animals showed higher serum levels of interleukin-10, suppression of interferon-gamma level, reduction of islet insulitis, and lower blood glucose levels compared to those treated with interleukin-10 plasmid alone or the nanoparticle alone up to week 6 post injection. Furthermore, it has been determined that Kir-GEM inhibits alpha ionic activity and prevents cell-surface expression of alpha subunits.
Histology of muscle showed that nanoparticles were biocompatible and did not cause a chronic inflammatory response. The investigators have proposed that in the presence of Ca2+, Kir-GEM binds to the beta isoform, and this interaction interferes in the trafficking or translocation of alpha subunits to the plasma membrane.
In addition to their use alone, delivery of both interleukin-4 and interleukin-10 DNA has also been investigated. Combined delivery into nonobese diabetic mice of the two plasmid DNA constructs encoding interleukin-4 and interleukin-10 (25 µg each) was done intravenously using a degradable, cationic polymeric carrier, poly (alpha-(4-aminobutyl)-L-glycolic acid)[27].
Novel cAMP signaling pathways of insulin releaseThe incretins are another set of factors that are important hormonal regulators of insulin secretion. Overexpression of the two cytokine messenger RNAs was confirmed in the liver of mice 5 days after delivery.
The incretins are polypeptide hormones released in the gut after a meal that potentiate insulin secretion in a glucose-dependent manner.
Six weeks after injection, 75% of observed islets were intact compared with less than 3% in the control group, and development of diabetes was prevented in 75% of treated animals at 30 weeks of age, compared to 20% in control mice receiving plasmid DNA coding for a single cytokine or vector control alone. Due to their dependence on ambient glucose for action, they are emerging as important new therapeutic agents to promote insulin secretion without accompanying hypoglycemia (a common complication of sulfonylurea treatment).Unlike sulfonylureas, incretins act by activating Gs (a G-protein that activates adenylyl cyclase) to increase cAMP in beta cells. The results indicated that the interleukin-4 and interleukin-10 plasmid DNAs had synergistic effects on the prevention of autoimmune diabetes. A report from the same research group showed that a ‘chimeric’ plasmid expressing both of the interleukin-4 and interleukin-10 under controls of two CMV promoters could also reduce insulitis in the same system [28].More recently, a research group also reported packaging plasmid DNA constructs coding for interleukin-4 and interleukin-10 into cationic nanomicelles to prevent type 1 diabetes [29]. Typically, the main mechanism of action of cAMP is by activation of an enzyme called protein kinase A (PKA) that, in turn, phosphorylates other substrates to turn on (or off) vital cell functions.
A single intramuscular dose of 50 microgram of the complex reduced levels of blood glucose and insulitis up to 6-week post delivery in 5-week-old streptozotocin-induced diabetic mouse.
Then, using molecular reagents that antagonize the effects of cAMPS, they observed that incretin-potentiated insulin secretion is attenuated. Notably, plasmid DNA coding for interleukin-4 and interleukin-10 has also been used as adjuvant to promote the therapeutic effect of DNA vaccines in a murine model for type 1 diabetes, which will describe later in this chapter in the ‘Glutamic Acid Decarboxylase DNA Vaccines’ section.In addition, a number of studies have reported that injection of plasmid DNA coding for cytokines normally considered pro-inflammatory can prevent diabetes. These results provide a mechanism whereby cAMP can directly promote exocytosis of insulin granules without activation of PKA (ie, a PKA-independent pathway), and thereby provide additional molecular targets for therapeutic intervention.7.
These results reflect the multifaceted role of cytokines on immune response [30], which could be dependent on dosage and time of administration. Beta cell dysfunction and apoptosisType one diabetes: Islet beta-cells are almost completely destroyed when patients with type 1 diabetes are diagnosed.
For example, administration of interleukin-18, also known as interferon-gamma inducing factor, can prevent diabetes in NOD mice [31, 32]. However, it was also shown that intramuscular electroporation of 2 x 100 microgram plasmid DNA coding for interleukin-18 into 4-6-week-old nonobese diabetic mice aggravates diabetes [33]. Another report showed that intraperitoneal administration of 30 microgram of plasmid DNA encoding interferon-gamma promotes insulitis in reovirus type-2 induced diabetic mice compared to controls [34].
In the absence of a defect in beta-cell function, individuals can compensate indefinitely for insulin resistance with appropriate hyperinsulinemia, as observed even in obese populations. This is in contrast with another report showing that injection of interleukin-12 induces interferon-gamma that prevents diabetes in NOD mice [35]. Together, these results suggest that there is risk associated with direct delivery of cytokines for the treatment of type 1 diabetes.
However, when allowance is made for the hyperglycaemia and the fact that glucose stimulates insulin secretion, it becomes apparent that the insulin levels in diabetic patients are lower than in healthy controls and inadequate beta-cell function therefore represents a key feature of the disease.
This possibility is suggested by a phase I clinical trial where new onset patients with type 1 diabetes received a combination treatment of interleukin-2 and the immunosuppressant rapamycin. Theoretically, the insulin secretory defect could result from either defects of beta-cell function or a reduction in beta-cell mass. Most quantitative estimates indicate that type 2 diabetes associates with either no change or < 30% reduction in beta-cell mass. Chemokine gene therapiesChemokines are a family of small chemotactic cytokines secreted by cells [36].
Moreover, the secretion defect is more severe than can be accounted for solely by the reduction in beta-cell mass.

Their name is derived from their ability to induce directed chemotaxis, or directed cell migration, in responsive cells. It therefore appears that the insulin secretory defect in type 2 diabetes does not primarily result from insufficient beta-cell mass but rather from an impairment of insulin secretion.8.
Some chemokines are considered pro-inflammatory and can be induced during an immune response to recruit cells of the immune system to a site of infection, while others are considered homeostatic and are involved in controlling the migration of cells during normal tissue maintenance and inhibiting abnormal inflammation like pathological autoimmune response. Prevention of beta cell dysfunction and apoptosis Islet beta-cells are almost completely destroyed when patients with type 1 diabetes are diagnosed.
Chemokines are involved in pathogenesis of autoimmune disease because they can selectively recruit various subsets of immune lymphocytes [37, 38]. Based on structural motifs near their N-terminal cysteine residue [C], chemokines are divided into four subfamilies, termed CXC, CX3C, C, and CC. The cure of type 1 diabetes requires beta-cell regeneration from islet cell precursors and prevention of recurring autoimmunity. The function of chemokines is modulated by the type of chemokine receptors they bind to as ligands on the surface of cells, and studies have shown that chemokines and chemokine receptors are involved in the pathogenesis of autoimmune diseases like type 1 diabetes.
Therefore, beta-cell replacement, regeneration and proliferation emerge as a new research focus on therapy for type 1 diabetes; however, its application is limited by the shortage of pancreas donors. Chemokine gene therapies for type 1 diabetes use anti-inflammatory chemokines as well as inhibitors of pro-inflammatory chemokine binding. In-vitro expansion of human cadaveric islet beta cells represents an attractive strategy for generation of abundant beta-like cells.
Human beta cells patent a very low proliferation capacity in vivo, and intact isolated islets cultured in suspension do not proliferate, although they remain functional for months. It was found that, in type 1 diabetic adult patients, elevated levels of serum CXC ligand-10 are associated with high-risk of disease in latent diabetic subjects [39].
When islets are allowed to attach, limited replication of beta cells can be induced by growth factors or extracellular matrix components before the beta-cell phenotype is lost. This finding was translated in animal models where blockade or neutralization of the CXC ligand-10 can prevent type 1 diabetes in nonobese diabetic mice [40].
Previous accepting of the determinants of tissue mass during adult life is still rudimentary. In virus-induced diabetic mice, virus infection results in rapid and differential expression of CXC receptor-3 and CXC ligand-10, which plays a dominant role in programming the ensuing autoimmune disease [41]. Insights into this problem may suggest novel approaches for the treatment of neoplastic as well as degenerative diseases. The blockade of CXC ligand-10 by using anti CXC ligand-10 monoclonal antibodies successfully aborts severity of antigen-specific injury of pancreatic beta cells and abrogates type 1 diabetes. In the case of the pancreas, elucidating the mechanisms that govern ? cell mass will be important for the design of regenerative therapy for both type 1 and type 2 diabetes, diseases characterized by an insufficient mass of ? cells. Mechanistically, the blockade impedes the expansion of peripheral antigen-specific T effector cells and hinders their migration into the pancreas.
It is clear that ? cell mass increase during pregnancy and in insulin-resistant states, but evidence on the ability of ? cells to regenerate from a severe, diabetogenic injury is conflicting.
A similar effect of the antibodies was confirmed in a cyclophosphamide accelerated model of type 1 diabetes [40]. Based on these reports, plasmid DNA encoding the CXC ligand-10 was constructed to induce production of anti-CXC ligand-10 antibodies in the host [42]. Igf1 has been shown to influence ?-cell apoptosis, and both Igf1 and Igf2 increase islet growth; Igf2 does so in a manner additive with fibroblast growth factor 2.
Intramuscular electroporation of 100 microgram of the plasmid DNA at 4 and 6 weeks of age induced synthesis of anti CXC ligand-10 antibodies in vivo, and suppressed the incidence of spontaneous diabetes which went from 75% in control mice down to 25% in treated mice at 30 weeks of age. Some study showed that IGF-1 can protect beta-cells from the destruction of apoptosis factors and promoting beta-cell survival and proliferation. Although this treatment did not inhibit insulitis or alter the immunological response, it enhanced the proliferation of pancreatic beta cells and resulted in an increase of beta-cell mass.A subsequent report from the same research group showed that combining complete Freund’s adjuvant with plasmid DNA encoding the CXC ligand-10 could reverse diabetes [43]. Interleukin-1beta (IL-1 beta) is a potent pro-inflammatory cytokine that has been shown to inhibit islet beta cell function as well as to activate Fas-mediated apoptosis in a nitric oxide-dependent manner.
Furthermore, this cytokine is effective in recruiting lymphocytes that mediate beta cell destruction in type one diabetes. Diabetes incidence was reduced from 70% in control mice to 20% in treated mice 10 weeks after plasmid DNA delivery.
IGF-I has been shown to block IL-1beta actions in vitro.Glucagon like peptide 1 (GLP-1) is a potent insulin secretagogue released by L-cells of the distal large intestine in response to meal ingestion and, together with glucose-dependent insulinotropic polypeptide (GIP), account for 90% of the incretin effect. In contrast, mice receiving complete Freund’s adjuvant and control plasmid DNA did not show disease reversal. In mice that were treated successfully, residual beta-cell mass was significantly increased, and some beta-cells were in a proliferative state.
GLP-1 also inhibits glucagon secretion, delays gastric emptying, and promotes weight loss by its appetite-suppressant effect. Although systemic cytokine profiles were unaffected, the frequency of regulatory T cells expressing CXC receptor-3 was significantly increased in local pancreatic lesions and possibly associated with the regulation of anti-islet autoimmunity. GLP-1 analogs also stimulate islet neogenesis and ?-cell replication and inhibit islet apoptosis. Another research group found that intra-pancreatic CC ligand-4 levels are increased in a model of diabetes protection by interleukin-4 treatment in female nonobese diabetic mice [44].
The gluco-incretin hormones GLP-1 and GIP can protect beta-cell against apoptosis induced by cytokines or glucose and free fatty acids.
The protective effect of CC ligand-4 was confirmed by abrogation of diabetes suppression after injection of anti-CC ligand-4 antibodies [45]. These result led to studies using CC ligand-4-encoded plasmid DNA therapy which showed that gene-gun delivery of 1 microgram of the plasmid DNA protects against type 1 diabetes in NOD mice, with diabetes rates reduced from 75% in control mice to 30% at 35 weeks of age when treated weekly from week 3 to 14, and from 80% in control mice down to 30% when treated weekly from week 9 to 14 [45]. Data also indicated that plasmid DNA delivery could both prevent and treat type 1 diabetes. Increases in cAMP levels, for instance as stimulated by GLP-1 or GIP action, potentiate glucose-stimulated insulin secretion by both protein kinase A (PKA)-dependent and independent mechanisms; they also stimulate gene transcription through PKA dependent phosphorylation of the transcription factor CREB.
This protection was associated with a T helper-2-like response in the spleen and pancreas, decreased recruitment of activated CD8 T cells to islets accompanied by diminished CC receptor-5 expression on CD8 T cells, and increased regulatory T cell activity in the draining pancreatic lymph nodes. To summarize, plasmid DNA encoding CC ligand-4 and CXC ligand-10 have been tested for their type 1 diabetic suppressive effects in spontaneous diabetic mouse models. Diabetes suppression is associated with decreased CD8 T lymphocyte activity and increased CD4 T regulatory cell activity.
Other cell-manipulating gene therapiesSeveral immune cell populations have deficiencies in type 1 diabetes, such as CD4 T lymphocytes, CD8 T lymphocytes, B lymphocytes, dendritic cells, macrophages, and NK cells in both nonobese diabetic mice and human patients [46, 47]. The goal of cell-manipulating gene therapy is to increase the diabetic suppressive function of cells like T regulatory or T helper-2 lymphocytes, which are considered important not only for therapeutic purposes, but also for playing a determining role in the development of type 1 diabetes.As mentioned at the beginning of this chapter, type 1 diabetes is a T helper-1-mediated autoimmune disease and strategies suppressing the function of these cells can be expected to have an impact on disease progression. Studies of mice with beta-cell specific inactivation of either receptor indicated that the insulin receptor was important for compensatory growth of the beta-cells in response to insulin resistance whereas the IGF-1 receptor was involved in the control of glucose competence. One of these strategies is the delivery of galectin-9, a carbohydrate-binding protein that regulates T helper-1 cells and induces their apoptosis through the galectin-9 receptor. Apoptosis, or programmed-cell-death, is a constantly ongoing process in steady state in vivo and helps maintain tissue and immune homeostasis. GLP-1 enhances beta cell function with an increase in the ability to secrete insulin and restore first phase insulin release. Mice treated with plasmid DNA coding for galectin-9 were significantly protected from diabetes: intravenous delivery of 2 x 100 microgram bi-weekly protected 85% of mice from diabetes versus 55% in controls [48]. Splenocytes from treated mice were also less responsive to mitogenic stimulation than splenocytes from the control group.
Within the pancreas, GLP-1 expands ?-cell mass via promotion of ?-cell growth and reduction of ?-cell death.?-Aminobutyric acid (GABA), a prominent inhibitory neurotransmitter, is present in high concentrations in ?-cells of islets of Langerhans. Data indicated that galectin-9 DNA may downregulate T helper-1 immune response in diabetic mice and could be used as a therapeutic agent in autoimmune diabetes.In contrast with galectin-9, decoy receptor 3 inhibits apoptosis. The GABA shunt enzymes, glutamate decarboxylase (GAD) and GABA transaminase (GABA-T) have also been localized in islet ?-cells.
The membrane protein is a member of the tumor necrosis factor receptor superfamily, and regulates immune responses by neutralizing apoptotic signals transmitted through CD95 (Fas receptor), lymphotoxin beta-receptor, and death receptor 3 on target cells. With the recent demonstration that the 64,000-Mr antigen associated with insulin-dependent diabetes mellitus is GAD, there isincreased interest in understanding the role of GABA in islet functions. As a result, transgenic expression of decoy receptor 3 in pancreatic beta cells protects nonobese diabetic mice from autoimmune diabetes [49].
Only a small component of ?-cell GABA is contained in insulin secretory granules, making it unlikely that GABA, co-released with insulin, is physiologically significant. When decoy receptor 3 is delivered systemically as plasmid DNA, it inhibits insulitis and diabetes by modulating immune responses.
Our immunohistochemical study of GABA in ?-cells of intact islets indicates that GABA is associated with a vesicular compartment distinctly different from insulin secretory granules. For example, four weekly intravenous injections of 100 microgram of plasmid DNA coding for decoy receptor 9 into nonobese diabetic mice was reported to reduce diabetes incidence from 90% in controls to 30% when treated at 4 weeks of age, 45% (started at 7 week old), and 70% (as Fc-fusion form, started at 12 week old) in 35-week-old female nonobese diabetic mice [50]. Treated mice showed less splenocyte proliferation and adoptive transfer of the cells ameliorated diabetes. Data also indicated that immune modulation by decoy receptor 3 may have been the result of differentiation and maturation of dendritic cells that subsequently regulated T effector differentiation and function.Cell migration is another process that plays a role in pancreatic beta cell destruction.
In this regard, plasmid DNA coding for CD44, which is a protein associated with cell migration and delivery of apoptotic signals by inflammatory cells, was investigated for the suppression of diabetes.
Physiological studies on the effect of extracellular GABA on islet hormonal secretion have had variable results. It was found that subcutaneous implants of a silicone tube filled with wound dressing sponge carrying CD44 encoded plasmid DNA could attenuate diabetes in a transfer model [51]. Diabetes was induced in male nonobese diabetic mice by transfer of diabetogenic splenocytes from female diabetic mice and was reduced from 90% in controls to 20-30% 12 weeks after two implants. The most compelling evidence for GABA regulation of islet hormone secretion comes from studies on somatostatin secretion, where it has an inhibitory effect. Here the mechanism of treatment was not thought to be strictly a gene therapy effect, but rather induction of anti-CD44 antibodies that inhibited CD44 function. Some researchers present new evidence demonstrating the presence of GABAergic nerve cell bodies at the periphery of islets with numerous GABA-containing processes extending into the islet mantle. An increasing body of evidence points to a possible relationship between the central nervous system and diabetes [52]. This close association between GABAergic neurons and islet ?- and ?-cells strongly suggests that GABA inhibition of somatostatin and glucagon secretionis mediated by these neurons. For example, the pancreatic autoantigen glutamic acid decarboxylase is an enzyme responsible for synthesis of the neurotransmitter gamma-aminobutyric acid (GABA) in the peripheral and central nervous system.
Notably, at least two neurotransmitter-related peptides have been used successfully as plasmid DNA immunotherapies for type 1 diabetes.
New evidence indicates that GABA shunt activity is involved in regulation of insulin secretion.
The first peptide is calcitonin gene-related peptide (CGRP), which has been found to play an important role in the regulation of T lymphocytes and in protecting cells from reactive oxygen species.
It was found that a single injection of 200 microgram plasmid DNA encoding the peptide delivered intramuscularly using electroporation could significantly ameliorate hyperglycemia and insulin deficiency [53]. These new observations provide insight into the complex nature of GABAergic neurons and ?-cell GABA in regulation of islet function. The treatment decreased diabetes incidence from 73% in controls to 23% at 28 days post delivery in a streptozotocin-induced diabetic model. Our study showed that GABA exerts has protective and regenerative effects on islet beta cells and reverses diabetes. The gene transfer also significantly inhibited T cell proliferation and secretion of the T helper-1 cytokine interferon-gamma, increased the levels of the T helper-2 cytokine interleukin-10, but had no effect on interleukin-4 and transforming growth factor-beta secretion. GABA therapy increased beta cell proliferation and decreased beta cell apoptosis, which in turn increase beta cell mass and induced the reversal of hyperglycemia in the different kind of mice. Therefore, calcitonin gene-related peptide gene transfer appears to ameliorate streptozotocin-induced diabetes through immune deviation.The second peptide is named vasoactive intestinal peptide (VIP) and functions as a neuromodulator and neurotransmitter [54]. Our data suggest that GABA exerts has ani-inflammatory effects, and is directly inhibitory to T cells and macrophages.Magnesium deficiency has recently been proposed as a novel factor implicated in the pathogenesis of the diabetic complications.
The peptide is a potent vasodilator that regulates smooth muscle activity, epithelial cell secretion, and blood flow in the gastrointestinal tract. Importantly, a body of evidence points to a significant role of vasoactive intestinal polypeptide in regulating immune responses. Photochemical analysis of those herbs have revealed the presence of flavonoids, which include quercetin and its derivatives.
The peptide acts as a potent endogenous anti-inflammatory molecule and promotes the activity of T regulatory cells, which makes it a promising candidate for the treatments of inflammatory and autoimmune diseases, such as septic shock, arthritis, multiple sclerosis, Crohn disease, and autoimmune diabetes [55, 56].
It is concluded that quercetin, a flavonoid with antioxidant properties brings about the regeneration of the pancreatic islets and probably increases insulin release in streptozocin-induced diabetic rats.Connective tissue growth factor (CTGF), to induce adult ? cell mass expansion. For example, a single intramuscular injection of 300 microgram of vasoactive intestinal polypeptide-encoding plasmid DNA significantly reduced the incidence of cyclophosphamide accelerated diabetes in female nonobese diabetic mice, from 70% in control to 30% on day 33 post delivery in 8-10-week-old mice [57]. Some study showed that CTGF is required for embryonic ? cell proliferation3, and that CTGF overexpression in embryonic cells increases ? cell proliferation and ? cell mass.
The mouse pancreas develops from ventral and dorsal evaginations of the posterior foregut endoderm at embryonic day, a process dependent on the transcription factors Pdx1 and Ptf1. Differentiation of all pancreatic endocrine cell types (?, ?, ? and PP) is dependent on the transcription factor, neurogenin 3 (Ngn3). Summary of Section 2Section 1 covers plasmid DNA encoding small protein molecules like cytokines, chemokines, peptides and other immune cell-manipulating agents with therapeutic effects on preclinical type 1 diabetes (Table 1).
Ngn3 expression is controlled by a variety of factors, including the Notch signaling pathway and the transcriptional regulators pancreatic and duodenal homeobox 1 (Pdx1), SRY-box 9 (Sox9) and hepatic nuclear factor 6 (Hnf6).
These approaches belong to systemic treatments and inevitably bear the risks associated with nonspecific immune suppression and chronic complications resulting from interference with the host immune system. Although ? cell neogenesis begins, these early insulin-positive cells do not contribute to mature islets. Nonetheless, if used as adjuvants or supplements to pancreatic autoantigen-targeting therapies like DNA vaccines, these approaches could be used selectively in DNA-based combination therapies.
Instead, endocrine cells that will go on to contribute to the mature islets begin to differentiate period known as the secondary transition. Although several factors have been identified that play a role in the regulation of embryonic and neonatal ? cell proliferation. One cell cycle regulator that does play a role in embryonic ? cell proliferation is the cell cycle inhibitor, p27Kip1. Inactivation of p27Kip1 during embryogenesis results in an increase in ? cell proliferation and subsequently ? cell mass. There was no change, however, in early postnatal ? cell proliferation, suggesting that p27Kip1 is not crucial to postnatal proliferation.As mentioned above Pdx1expressed in multipotent pancreatic progenitors in the early stages of pancreas development, but, Pdx1 expression becomes enhanced in insulin-positive cells and is found at only low levels in exocrine cells. This expression pattern is maintained into adulthood and Pdx1 plays a critical rolein maintenance of the mature ? cell phenotype. Inactivation of Pdx1 in embryonic insulin-expressing cells results in a dramatic decrease in ? cell proliferation at late gestation, leading to decreased ? cell mass at birth and early onset diabetes.